Long-chain fatty acid (LCFA) oxidation has been shown to play an important role in interleukin-4 (IL-4)-mediated macrophage polarization (M(IL-4)). However, many of these conclusions are based on the inhibition of carnitine palmitoyltransferase-1 with high concentrations of etomoxir that far exceed what is required to inhibit enzyme activity (EC < 3 μM). We employ genetic and pharmacologic models to demonstrate that LCFA oxidation is largely dispensable for IL-4-driven polarization. Unexpectedly, high concentrations of etomoxir retained the ability to disrupt M(IL-4) polarization in the absence of Cpt1a or Cpt2 expression. Although excess etomoxir inhibits the adenine nucleotide translocase, oxidative phosphorylation is surprisingly dispensable for M(IL-4). Instead, the block in polarization was traced to depletion of intracellular free coenzyme A (CoA), likely resulting from conversion of the pro-drug etomoxir into active etomoxiryl CoA. These studies help explain the effect(s) of excess etomoxir on immune cells and reveal an unappreciated role for CoA metabolism in macrophage polarization.
SUMMARY Upon antigen recognition and co-stimulation, T lymphocytes up-regulate the metabolic machinery necessary to proliferate and sustain effector function. This metabolic reprogramming in T cells regulates T cell activation and differentiation but is not just a consequence of antigen recognition. While such metabolic reprogramming promotes the differentiation and function of T effector cells, the differentiation of regulatory T cells employs different metabolic reprogramming. We therefore hypothesized that inhibition of glycolysis and glutamine metabolism might prevent graft rejection by inhibiting effector generation and function and promoting regulatory T cell generation. We devised an anti-rejection regimen involving a glycolytic inhibitor, 2-Deoxyglucose (2-DG), an anti-Type II diabetes drug (metformin) and an inhibitor of glutamine metabolism, 6-Diazo-5-oxo-L-norleucine (DON). Using this triple drug regimen we were able to prevent/delay graft rejection in fully mismatched skin and heart allograft transplantation models.
Mitochondrial homeostasis vitally depends on mitophagy, the programmed degradation of mitochondria. The roster of proteins known to participate in mitophagy remains small. We devised here a multidimensional CRISPR/Cas9 genetic screen, using multiple mitophagy reporter systems and pro-mitophagy triggers, and uncover numerous new components of Parkin-dependent Reprints and permissions information is available at www.nature.com/reprints.Users may view, print, copy, and download text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use:
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